| Budget Amount *help |
¥3,890,000 (Direct Cost: ¥3,800,000、Indirect Cost: ¥90,000)
Fiscal Year 2007: ¥390,000 (Direct Cost: ¥300,000、Indirect Cost: ¥90,000)
Fiscal Year 2006: ¥300,000 (Direct Cost: ¥300,000)
Fiscal Year 2005: ¥3,200,000 (Direct Cost: ¥3,200,000)
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| Research Abstract |
Entrapment of proteins in wet, optically transparent, porous silica gel matrices has enabled a dramatic expansion of the folding time, allowing direct observation of the entire folding pathway using spectroscopic techniques. In this study, we have focused on the folding reactions of the following three model proteins.
1. Horse cytochrome c
In wet silica gels, cytochrome c folds over a period of hours, days, weeks, or longer, depending on the aging time. During refolding in silica gels, collapse and helix formation occur in a stepwise manner, as observed in aqueous solution. Analysis of kinetics and transient spectra reveals a sequence of four distinct intermediates with progressively increasing degree of folding, two of which closely resemble those previously characterized in solution, namely, the early collapsed and the molten globule intermediates. The other two are the pre-collapsed and pre-molten globule intermediates that may escape detection by conventional kinetic methods.
2. Bovine ubiquitin
While ubiquitin folds in an apparent two-state manner in aqueous solution, a partially structured intermediate is populated on the folding pathway of ubiquitin when entrapped in wet silica gels. This finding suggests an interesting possibility that our approach is able to stabilize and capture on-pathway "hidden" folding intermediates that may exist on the folding pathway but escape detection by conventional techniques.
3. Bovineβ-lactoglobulin
The refolding of β-lactoglobulin was also very slow in wet silica gels. Analysis of the transient CD spectra allows us to probe the early folding events that consist of non-native a-helix formation, native β-sheet core formation, and further a-helix to (β-sheet transition. These results illustrate the potential usefulness of our approach for high-resolution analysis of rapid folding events without significantly perturbing the folding pathway.
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